This project is aimed at delineating specific molecular mechanisms by which signals received by immune cells result in activation of genes critical to fighting diseases. In particular, we have focused on the activation of the family of transcription factors collectively referred to as NF-kappaB, because they are essential for inducing the expression of many important immune effectors. In addition, NF-kappaB factors are essential to expression of the HIV as well as several other viruses of clinical relevance. This project focuses on the transcription factor-proximal events which occur during signal-induced activation. The activation process proceeds via inactivation of inhibitors (IkappaBs) of the transcription factors, which allows the then liberated NF-kappaB complexes to travel from the cytoplasm into the nucleus, where they exert their effects. An understanding of the molecular details of the inactivation of these inhibitors is fundamental to understanding immune activation in a variety of cells and it is likely to provide the basis to understanding diseases in which immune activation is uncontrolled or defective. In addition, such knowledge will identify potential new targets for therapeutic intervention in inflammatory diseases as well as viral diseases in which NF-kappaB plays a critical role, such as AIDS. We have previously demonstrated that signal-induced activation of the NF-kappaB transcription factors involves rapid site-specific phosphorylation followed by proteolytic degradation of the primary cytoplasmic inhibitor, IkappaB-alpha. Degradation is carried out by proteasomes in a ubiquitin-dependent fashion. We have also determined that the inhibitor becomes ubiquitinated on specific lysines as a consequence of induced phosphorylation; the ubiquitinated species is then degraded by proteasomes. The phosphorylation and ubiquitination sites are located near the N-terminus of IkappaB-alpha. We have now shown that efficient degradation of IkappaB-alpha also requires PEST sequences located near its C-terminus. We demonstrate that the short N- and C-terminal domains of IkappaB-alpha are not only necessary but also sufficient to confer an inducible degradation phenotype; attachment of these two domains to a completely unrelated protein causes signal-induced degradation of the chimeric protein. This research suggests a novel way to regulate similarly designed chimeric proteins in cells. We have established in vitro systems in which IkappaB-alpha phosphorylation is induced in cellular extracts by addition of specific kinases which initiate an as yet unknown cascade of events. These systems permit further dissection to identify the molecular components involved in transmitting signals to IkappaB-alpha, including the IkappaB-alpha kinase itself. Identification of such a kinase or its direct activators are of great interest as they should provide ideal targets for possible therapeutic intervention aimed at stopping undesirable immune activation and inflammation. Our in vitro systems also permit us to dissect the molecular requirements for ubiquitination of phosphorylated IkappaB-alpha, and in particular they will provide the basis for identification of the ligase component which specifically recognizes this modified form of IkappaB-alpha.